R.A. Donadelli, J.G. Pezzali, P.M. Oba , C. Pendlebury ... · and Aldrich, 2019; Pezzali et al.,...

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© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

A commercial grain-free diet does not decrease plasma amino acids and taurine status, but

increases bile acid excretion when fed to Labrador Retrievers

R.A. Donadelli*, J.G. Pezzali*, P.M. Oba†, K.S. Swanson†, C. Coon‡, J. Varney‡, C. Pendlebury§, A.K.

Shoveller#

* Animal Biosciences Department, University of Guelph, ON, Canada.

† Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL.

‡ Department of Poultry Science, University of Arkansas, Fayetteveille, AR.

§ Champion Petfoods LP, Edmonton, AB, Canada.

# Animal Biosciences Department, University of Guelph, ON, Canada, 50 Stone Road East, Guelph,

Ontario, Canada, N1G 5L3, email: [email protected]

Acknowledgements

Champion Petfoods LP freely shared data from the AAFCO study reported herein. C.C. and C.P.

designed research. C.C. conducted research, and all authors analyzed the data, wrote the paper, and

had responsibility for final content. All authors read and approved the final manuscript.

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ic.oup.com/tas/article-abstract/doi/10.1093/tas/txaa141/5875988 by guest on 27 July 2020

mailto:[email protected]

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ABSTRACT: Grain-free diets tend to have greater inclusions of pulses in contrast to grain-based diets.

In 2018 the Food and Drug Administration (FDA) released a statement that grain-free diets may be

related to the development of canine dilated cardiomyopathy (DCM). However, all dog foods met

regulatory minimums for nutrient inclusion recommended by the Association of American Feed

Controls Official (AAFCO). In some FDA case reports, but not all, dogs diagnosed with DCM also had

low concentrations of plasma or whole blood taurine, as such we hypothesized that feeding these

diets will result in reduced taurine status from baseline measures. The objective of this study was to

determine the effects of feeding a grain-free diet to large breed dogs on taurine status and overall

health. Eight Labrador Retrievers (4 males, 4 females; Four Rivers Kennel, MO) were individually

housed and fed a commercial complete and balanced grain-free diet (Acana Pork and Squash

formula; APS) for 26 wk. Fasted blood samples were collected prior to the start of the trial (baseline;

wk 0), at wk 13 and wk 26 for analyses of blood chemistry, hematology, plasma amino acids (AA),

and whole blood taurine. Urine was collected by free catch at wk 0 and 26 for taurine and creatinine

analyses. Fresh fecal samples were collected at wk 0 and 26 for bile acid analyses. Data were

analyzed using the GLIMMIX procedure with repeated measures in SAS (v. 9.4). Plasma His, Met, Trp,

and taurine and whole blood taurine concentrations increased over the course of the study (P <

0.05). Urinary taurine to creatinine ratio was not affected by diet (P>0.05). Fecal bile acid excretion

increased after 26 wk of feeding APS to dogs. Despite the higher fecal excretion of bile acids, plasma

and whole blood taurine increased over the 26-wk feeding study. These data suggest the feeding

APS, a grain-free diet, over a 26-wk period improved taurine status in Labrador Retrievers and is not

the basis for incidence of DCM for dogs fed APS. Other factors that may contribute to the etiology of

DCM should be explored.

Keywords: Grain-free dog food, Large breed dogs, Pulses, Sulfur amino acids.

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INTRODUCTION

In 2018, the Food and Drug Administration (FDA) released a report warning about a possible

link between feeding dogs (specifically large male dogs) grain-free diets and the development of

dilated cardiomyopathy (DCM); efforts to understand the potential link continues. Grain-free diets

are loosely defined as dog foods which contain no grains and instead contain peas, lentils, other

leguminous seeds (pulses), and/or potatoes in various forms (whole, flour, protein, etc.) as a main

ingredient (listed within the first 10 ingredients in the ingredient list, before vitamins and minerals).

Legumes, specifically pulses, have greater protein (NRC, 2006; Singh, 2017) and fiber (especially

soluble fibers and oligosaccharides) content (Carciofi et al., 2008) in contrast to grains (Alvarenga

and Aldrich, 2019; Pezzali et al., 2020). While these characteristics may help to increase dietary

protein content, and aid in gut health and weight management (Choct, 2009; German et al., 2010;

Flanagan et al., 2017; Kroger et al., 2017), pulses contain lower concentrations of sulfur amino acids

(SAA) and may cause imbalances in a diet’s amino acid profile. Other limitations in pulses nutrient

composition include the complete lack of taurine and L-carnitine content (NRC, 2006; Hall et al.,

2017; Mansilla et al., 2019) which are required to support osmoregulatory balance and fatty acid

oxidation, repectively.

There is a known relationship between low taurine status and DCM. One common parameter

reported to the FDA was low plasma and whole blood taurine concentrations in dogs with DCM. In

Newfoundland dogs with DCM, taurine supplementation was reported to reverse the condition

(Fascetti et al., 2003). Additionally, when taurine was supplemented to Golden Retrievers with DCM,

there was an improvement in health, and in some cases the disease was resolved (Belanger et al.,

2005). Similarly, other cases of taurine supplementation in DCM-diagnosed dogs lead to the similar

outcomes (Kittleson et al., 1997; Backus et al., 2006; Kaplan et al., 2018; Adin et al., 2019).

Taurine is a β-amino amino sulfone that is not used for protein synthesis, but remains in

animal tissues as a free AA (Brosnan and Brosnan, 2006). Dogs are able to synthetize taurine if

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sufficient dietary SAA (Met and cyst(e)ine) are provided (Torres et al., 2003). As a result, despite

being involved in several metabolic roles (Huxtable, 1992; Sanderson, 2006), taurine is not

considered an essential nutrient for dogs (NRC, 2006; AAFCO, 2018). One of the primary roles of

taurine in dogs is the conjugation of bile acids (BA, Czuba and Vessey, 1981). After primary BA aid in

fat digestion, then they are re-absorbed and transported back to the liver (Dawson and Karpen,

2015); however, some dietary components may affect BA recirculation. For example, BA

recirculation can be affected by fibers (Kritchevsky, 1978) and high fat content (Bravo et al., 1998;

Herstad et al., 2017; Herstad et al., 2018). Since taurine is conjugated to BA, loss could occur through

feces due to reduced enterohepatic recirculation of BA (Hickman et al., 1992; Ajouz et al., 2014;

Dawson and Karpen, 2015) and result in a reduction in taurine status. Moreover, taurine is also lost

through urinary excertion, with dietary supplementation causing an increase in excretion (Park et al.,

1989). As such, taurine balance can be assessed by measuring plasma and whole blood taurine, fecal

BA excretion, and urinary taurine excretion.

To date, there are few published studies evaluating the effects of a commercial grain-free diet

and taurine status in healthy large-breed dogs, and most pet food companies do not publish AAFCO

feeding studies. Thus, the objective of this study was to evaluate the effects of feeding a commercial

grain-free dog food to Labrador Retrievers on apparent total tract digestibility (ATTD), stool quality,

blood chemistry, hematology, plasma AA and taurine concentrations and BA excretion during a 26-

wk feeding trial that followed AAFCO Canine Feeding protocols. We hypothesized that dogs fed a

commercial complete and balanced grain-free diet with adequate concentrations of SAA and taurine

would: 1) have normal blood chemistry and hematology; 2) normal plasma AA concentrations; 3)

reduced blood and plasma taurine concentrations; and 4) a higher fecal excretion of primary and

secondary BA and urinary taurine.

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MATERIALS AND METHODS

The Institutional Animal Care and Use Committee (IACUC) of Four Rivers Kennel (Walker, MO)

approved this study (IACUC number FRK-14). The study was conducted at Four Rivers Kennel from

October 18, 2018 to April 18, 2019. The study used monadic feeding with repeated measures and

followed the recommendations of the AAFCO (2018) feeding protocol to support a complete and

balanced adult maintenance claim. Additional outcomes focused on taurine metabolism were added

to test the hypotheses.

Dog food and feeding trial

Eight Labrador Retriever dogs (4 intact males and 4 intact females, average age = 6.0 ± 2.1 yr)

were housed individually (1.83m x 1.22m) and fed a grain-based commercial control diet (CTL; MFA

Gold N Pro, MFA Inc., Columbia, MO, USA; Table 1) for 26 wk washout prior to the start of the study.

Kennel temperature was held between 13ºC and 29ºC and humidity was dependent on the weather.

Depending on weather conditions, dogs had socialization with other dogs for up to 6 h daily in

outside runs (12.2m x 12.2m).

After the 26 wk washout period (baseline; wk 0), dogs were fed the treatment grain-free diet

(APS; Acana Pork and Squash formula, Champion Petfoods, Auburn, KY, USA). Males were fed 700

g/d and females were fed 550 g/d; feeding amounts were based on their energy requirements from

the previous 26 wk. Clean, fresh water was available ad libitum. Any remaining food was weighed

daily to calculate total feed intake.

Blood collection and analyses

At wk 0, 13, and 26, blood samples were collected between 9:00 and 10:00 am after dogs

were fasted for 24 h. Blood was sampled using two lithium heparin tubes (4 mL per tube; BD

367884) and one EDTA tube (2 mL; BD 367841). After blood collection, tubes were gently inverted 8

to 10 times and stored in a refrigerator (approximately 2ºC) before analysis. One of the lithium

heparin tubes from each dog was shipped with cool packs to the University of California, Davis

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Amino Acid Laboratory (Davis, CA) for whole blood taurine analysis. The other two lithium heparin

tubes were centrifuged at 1500 x G for 10 min at 4ºC and the plasma (approximately 1.2 mL) was

transferred to a cryovial. One of the cryovials was shipped to the University of California, Davis

Amino Acid Laboratory (Davis, CA) for the determination of plasma amino acids according to Kim et

al. (1995). All samples that were shipped to a laboratory were sent with cool packs and were

received by the laboratories on the next day to decrease sample degradation. Another 0.25 mL of

plasma was collected and refrigerated for blood chemistry analysis (Abaxis VetScan2) at the Four

Rivers Kennel Laboratory (Walker, MO). The EDTA tube was stored refrigerated (2ºC) until blood

hematology was determined at Four Rivers Kennel Laboratory using an Abaxis HM5 (Abaxis, Union

City, CA, USA).

Fecal and urine collection and analyses

During the 5-d fecal collection phase, feces were scored in the morning of each collection day

on 5-point scale (1 being liquid diarrhea and 5 being feces that are fully formed, very firm, with little

moisture, hard and crumbles easily) and collected in a specimen cup and frozen at -20ºC. After the 5

d of collection the content of the specimen cups for each dog was homoginized using a hand blender

and kept frozen. A subsample was sent to Eurofins Scientific Inc. (Des Moines, IA, USA) to be

analyzed for dry matter and macronutrient concentration. Urine samples collected once for each

dog during the 5 d of fecal collection before feeding by free catch in a clean ladle. After being

collected, the urine samples were refrigerated (2ºC) for approximately 1 h before they were

centrifuged at 1000 x G for 20 min at 4ºC. After centrifugation, 1-mL aliquot was separated and

analyzed for taurine (University of California Davis Amino Acid Laboratory) and a 3-mL aliquot for

creatinine (University of California Davis Central Laboratory) concentrations. In addition, a fresh fecal

sample (approximately 20 g) were collected and frozen at -80ºC at baseline and wk 26 for

subsequent fecal BA analyses (Gastrointestinal Laboratory, Texas A&M University, College Station,

TX).

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Apparent total tract macronutrient digestibility estimation

During the last 6 d of the feeding trial, all dogs received a capsule containing 2 g of titanium

dioxide with their morning feeding for the estimation of apparent total tract macronutrient

digestibility. Fecal samples were collected during the last 5 d of wk 26, homogenized, and frozen

prior to other sample preparation. Fecal samples were analyzed for moisture (AOAC 930.15), crude

protein (AOAC 990.03), acid-hydrolyzed fat (AOAC 954.02), ash (AOAC 942.05), neutral detergent

fiber (Ankom NDF), acid detergent fiber (Ankom ADF 05-03), and titanium dioxide (Myers et al.,

2004; Alvarenga et al., 2019). ATTD was estimated using the equation below:

(

)

wherein: TD is the titanium dioxide content in the diet as a percentage, NF is the nutrient

concentration in the feces as a percentage, TF is the titanium dioxide content of the feces as a

percentage, and ND is the nutrient concentration in the diet as a percentage.

Statistical analysis

This study was designed as a monadic feeding study and data were analyzed as repeated

measures with time considered as a fixed effect. The GLIMMIX procedure (SAS v. 9.4, The SAS

Institute, Cary, NC, USA) and a repeated measures model were used to analyze the data. Dog was

considered the experimental unit and dog nested within wk was considered the random effect.

Means were separated by Fisher’s LSD considering an alpha of 0.05. Data reported in tables are

expressed as least square means (± standard error of the mean).

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RESULTS AND DISCUSSION

The current study sought to describe the physiological effects of feeding a commercial grain-

free formula on taurine status and overall health of Labrador Retriever dogs. Due to the FDA reports

on the possible association between increased presentation of DCM cases and diet fed to those

dogs, this study collected key data surrounding SAA metabolism and specifically taurine. The dogs

started the trial with plasma Met concentrations above the upper limit of the reference range (93.5

nmol/mL, range 57 ± 2 nmol/mL; Table 2), yet plasma Met concentrations increased over time (P <

0.05) and plasma cystine was not affected (P > 0.05). This suggests that diet and protein turnover

may have maintained plasma Met concentrations. Met is used as a methyl donor, in the production

of Cys and other sulfur-containing compounds. Accurate quantification of total Cys requires

biological samples to be reduced and prepared differently than methodology used to measure the

other AA (Shoveller et al., 2003; Shoveller et al., 2004). Because the analyses used does not quantify

total cyst(e)ine and cystine is not considered an accurate measure of total cyst(e)ine we have chosen

not to discuss these results. Additionally, Cys is known to be reactive and losses occur rapidly after

blood is collected (Torres et al., 2004). In the present study, samples were shipped overnight to UC

Davis Amino Acid Laboratory in cool packs; however, this may not have been enough to prevent

sample degradation and it is a limitation of this work. Thus, Cys and cystine values here reported

must be considered with caution. Future studies should consider different methods to measure total

cyst(e)ine (Torres et al., 2004) and also quantify homocysteine and glutathione to better understand

the regulation of SAA metabolism in dogs.

Whole blood concentrations of taurine tend to be more accurate and is an indication of

intracellular taurine content (Pacioretty et al., 2001) in contrast to plasma concentrations. One

possible explanation for theses differences is that taurine can be released from white blood cells and

platelets during clotting (Torres et al., 2004; Kaplan et al., 2018); therefore, plasma taurine content is

more variable than in whole blood. In the current study, whole blood taurine concentrations were

above the upper confidence limit of 250 nmol/mL (Kaplan et al., 2018) only at wk 26. This is an

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indication that whole body taurine status increased in Labrador Retriever dogs consuming APS

(Table 2). The increase in taurine over time may be due to the higher concentrations of taurine in

the APS diet compared to the CTL (0.14 vs. 0.07, respectively). Similarly, Pezzali et al. (2020) reported

an increase in plasma taurine concentration in Beagle dogs fed a grain-free diet supplemented with

taurine over a 28-d feeding trial. Although whole blood and plasma concentrations of taurine are

commonly used to evaluate taurine deficiency in dogs in a clinical setting, it is as important to

consider the urinary and fecal losses. Whole body taurine pool size can be regulated through urinary

reabsorption/excretion (Chesney et al., 2010). Urinary concentrations of taurine, presented as

taurine:creatinine, were similar (P > 0.05) between wk 0 and wk 26 and suggests that dogs were not

taurine deficient (Table 3). In contrast, Pezzali et al. (2020) reported that Beagle dogs fed a grain-free

diet and supplemented with taurine had a higher urinary taurine:creatinine excretion at d 14 and 28

compared to baseline measurements. This difference could be related to the higher taurine

concentration of the diets fed in the study by Pezzali et al. (2020) compared to our diets (0.34% vs.

0.14%, respectively), but suggests that both grain-free diets met or exceeded the metabolic

requirements for taurine.

In addition to urinary losses, fecal loss of taurine occurs when the BA are not reabsorbed

through the enterohepatic circulation (Hickman et al., 1992; Ajouz et al., 2014; Dawson and Karpen,

2015). In the dog, most of the primary BA (cholic acid and chenodeoxycholic acid) are synthesized

from cholesterol and conjugated with taurine (Czuba and Vessey, 1981; Imamura et al., 2000), rather

than glycine (Zhang et al., 2016) before being secreted into the small intestine. Hamsters who

consume greater dietary taurine experienced increased BA secretion (Bellentani et al., 1987); while

this is yet to be measured in dogs, dietary taurine supplementation could also lead to an increase in

BA secretion in dogs; however, it is generally accepted that the supplementation of taurine will lead

to excretion in the urine (Chesney et al., 2010). If the BA are not reabsorbed, taurine can be

deconjugated by the commensal bacterial enzyme BA hydrolase present in the terminal ileum and

colon (Long et al., 2017). The unconjugated primary BA can be converted into secondary BA by

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Clostridium (cholic acid converted to deoxycholic acid), Eubacterium (chenodeoxycholic acid

converted to lithocholic acid and ursodeoxycholic acid), and other bacterial taxa present in the

colon, through the enzyme 7 α-dehydroxylase (Doerner et al., 1997; Benno et al., 2001; Ridlon et al.,

2006). As such, fecal concentrations of BA may be used as a tool to evaluate fecal losses of taurine

(Anantharaman-Barr et al., 1994).

In the current study, fecal concentrations of total BA increased from baseline, and at wk 26

were 1.56 times greater (Table 3). This was due to a higher (P < 0.05) excretion of both primary (wk 0

= 0.063 vs. wk 26 = 0.179 µg/mg) and secondary (wk 0 = 4.19 vs. wk 26 = 6.64 µg/mg) BA. The

increase in primary BA was a result of greater excretion (P < 0.05) of both cholic acid (wk 0 = 0.055

vs. wk 26 = 0.142 µg/mg) and chenodeoxycholic acid (wk 0 = 0.008 vs. wk 26 = 0.037 µg/mg). Pezzali

et al. (2020) also observed a greater excretion of primary BA due to the excretion of cholic acid in

Beagle dogs fed a grain-free diet; however, the excretion of chenodeoxycholic acid was not a factor.

The excretion of BA can be affected by diet (Hestad et al., 2018); thereby, some dietary components

(e.g., dietary fibers and fats) in the APS diet may play a role in the excretion of BA. The higher fecal

primary BA concentrations after 26 wk of APS consumption may be due to the higher dietary fat

content of the APS diet (wk 0 = 16.8% vs. wk 26 = 18.8%; Table 1), which would increase BA

secretion (Bravo et al., 1998). An increase in BA excretion has been previously reported when dogs

were fed a high minced beef diet compared to a commercial dry food as a response to different

dietary fat levels (33.1% vs. 16.33%, respectively; Herstad et al., 2017; Herstad et al., 2018).

In addition to the dietary fat content, dietary fibers may also bind BA in the small intestine and

further increase BA excretion (Kritchevsky, 1978). Moreover, the fermentation of dietary fibers in

the large intestine, may lower the luminal pH and bacterial 7 alpha-dehydroxylase activity, reducing

the conversion of primary BA to secondary BA (Bingham, 2000). Unfortunately, products of

fermentation (e.g., short-chain fatty acids) and fecal pH were not analyzed in the present study and

should be considered in future research. Despite the TDF of APS being less than CTL (11.4% vs.

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13.6%; Table 1), the oligosaccharide content (not measured), likely was higher in APS, due to the

higher inclusion of pulses (Bednar et al., 2001; de Oliveira et al., 2012). Dietary oligosaccharide

content contributes to an increase in bacterial fermentation in the gut (Felix et al., 2013). Ko and

Fascetti (2016) also reported a higher excretion of BA when dogs were fed a purified diet with beet

pulp (a moderately fermentable fiber). Although both this study and Ko and Fascetti (2016) reported

low soluble fiber content for the experimental diets, there was an increase in BA excretion when

dogs were fed APS for 26 wk. However, it is noteworthy that both plasma and whole blood taurine

concentrations were improved over time for dogs fed APS and urinary taurine:creatinine was not

affected by the diet. Therefore, even though greater fecal losses of BA occurred, this did not affect

taurine status as supported by plasma and whole blood taurine concentrations and urinary taurine:

creatinine throughout the duration of the study.

Secondary BA also increased from the baseline (wk 0 = 4.19 vs. wk 26 = 6.46 µg/mg), where

deoxycholic acid was the major contributor (P< 0.05; wk 0 = 3.45 vs. wk 26 = 5.52 µg/mg). The rise in

deoxycholic acid concentration, rather than that of lithocholic acid and ursodeoxycholic acid,

indicates a greater ability of gastrointestinal bacteria to transform cholic acid rather than

chenodeoxycholic acid. This could be due to substrate (cholic acid vs. chenodeoxycholic acid)

availability, microbial populations present in the colon, or other unknown factors. Likewise, in a

previous study, secondary BA and deoxycholic acid concentrations increased after dogs consumed a

high minced beef diet for 7 wk compared to dogs fed a commercial dry food (Herstad et al., 2018).

Unfortunately, microbiota population shifts due to the consumption of the grain-free diet were not

analyzed in the present study. However, our results are different from Pezzali et al. (2020), who did

not report a greater excretion of secondary BA due to deoxycholic acid in Beagle dogs fed a grain-

free diet. Thus, a combination of factors in the APS diet, such as higher dietary fat content and

soluble fiber concentrations, may have stimulated the higher concentrations of both fecal primary

and secondary BA (Table 3).

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We also observed a decrease in ursodeoxycholic acid and lithocholic acid as percentage of

total BA [ursodeoxycholic acid: wk 0 = 0.2% vs. wk26 = 0.1% (p = 0.02); lithocholic acid: wk0 = 18.1%

vs. wk26 = 14.7% (p = 0.04)]. In addition to BA concentration, primary and secondary BA as

proportion of total BA were not affected over time (P > 0.05; Table 3). The level of hydrophobicity of

BA is positively associated with its cytotoxic potential (BA hydrophobicity scale: ursodeoxycholic acid

< cholic acid < chenodeoxycholic acid < deoxycholic acid < lithocholic acid; Hofmann,1999), with

deoxycholic acid and lithocholic acid known to induce oxidative damage of DNA in vitro (Booth et al.,

1997; Bernstein et al., 1999; Glinghammar, 2002; Payne, 2008; Rosignoli et al., 2008). In contrast,

ursodeoxycholic acid is believed to have chemoprotective potential (Alberts et al., 2005; Akare et al.,

2006). For this reason, a lower percentage of fecal lithocholic acid after 26 wk of consumption of APS

may suggest a beneficial shift. However, the reduction in fecal ursodeoxycholic acid percentage may

not be desirable. Our results are different from Pezzali et al. (2020) who observed a greater

excretion of primary BA as a proportion of total BA and a lower excretion of secondary BA

(deoxycholic acid + lithocholic acid) in Beagle dogs fed a grain-free diet. However, similar to the

present study Pezzali et al. (2020) also reported a lower excretion of lithocholic acid as a proportion

of total in Beagle dogs fed a grain-free diet (information regarding ursodeoxycholic acid was not

reported).

Fasting plasma concentrations (24 hr fast) of Arg, Ile, Thr, and Val did not change over the

course of the trial (P > 0.05; Table 2). In contrast, plasma concentrations of His, Leu, Lys, Met, Phe,

and Trp increased from wk 0 to wk 26 (P < 0.05; Table 2), suggesting these AA were over

requirement and drove increased protein turnover. Plasma AA concentrations are generally tightly

controlled, increasing after consuming a meal and subsequently decreasing to fasting

concentrations. Concentrations of Asp, 3-methyl-L-histidine, Leu and Lys increased over time (P <

0.05), suggesting that there was greater protein turnover as the trial progressed, and increasing the

rate of protein turnover. This is supported by the increase in 3-methyl-L-histidine, which is a marker

for protein breakdown (Chinkes, 2005; Holm and Kjaer, 2010) and is further supported by the

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increase over time of blood urea nitrogen, which is an indication of AA catabolism and subsequent

ureagenesis. Blood urea nitrogen is associated with dietary protein and is likely due to the greater

protein content of the test diet as compared to the baseline diet. Increased circulating indispensable

AA and blood urea nitrogen are positively related to dietary protein intake (Kang et al., 1987). Future

studies should consider dynamic measures of protein synthesis, such as rates of protein synthesis, or

static measures over time, such as body composition by DXA or qMRI, to more accurately reflect the

impact of AA status of dogs fed grain-free formulas.

Despite differences in plasma AA and fecal BA, during the study, the stool of dogs fed APS

were considered ideal, with an average fecal score of 3.12 ± 0.13. Dry matter, crude protein, and

crude fat apparent total tract digestibility were 86.6%, 90.5%, and 95.4%, respectively. The current

study observed similar digestibility values as compared to Pezzali and Aldrich (2019; dry matter:

86.6% vs. 85.8%; crude protein: 90.5% vs. 87.2%; crude fat: 95.4% vs. 93.6%, respectively) who also

fed a grain-free diet to dogs (38.0% crude protein, 12.5% crude fat, 3.85% insoluble fiber, 6.22%

soluble fiber, 4.33% ash; ingredients: hydrolyzed pork protein, white potato, green peas, tapioca

starch, minerals and vitamins, menhaden fish oil, taurine, antioxidant, chicken fat, flavor enhancer,

and titanium dioxide) for a period of 28 d. Small differences between these two studies may be

related to differences in breed (Labrador Retrievers vs. Beagles), environment, analytical variation,

ingredient and nutrient composition.

In the present study, dry matter and crude protein digestibilities were higher (dry matter:

86.6% vs. 79.6%, crude protein: 90.5% vs. 85.3%, respectively for this study and Chiofalo et al., 2019)

than what Chiofalo et al. (2019) reported for Labrador Retrievers fed a grain-free diet (39.24% crude

protein, 18.69% crude fat, 11.59% TDF, 7.91% ash; ingredients: fresh grass fed lamb, dehydrated

lamb meat, potatoes, dried whole eggs, fresh herrings, dehydrated herring, chicken fat, herring oil,

vegetable pea fiber, dried carrots, sun-cured alfalfa meal, inulin, fructooligosaccharinde, mannan-

oligosaccharides, dehydrated blueberry, dehydrated apple, dehydrated pomegranate, dehydrated

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sweet orange, dehydrate spinach, psyllium seed husk, currant powder, salt, brewers dried yeast,

turmeric, glucosamine, chondroitin sulfate, tagete flower extract). Similar to Chiofalo et al. (2019),

Meyer and others (1999) reported lower values for crude protein and fat digestibilities (86.3% and

93.6%, respectively) in Labrador Retrievers fed a commercial diet (29.4% crude protein, 13.8 crude

fat, 2.84% insoluble fiber, 0.55% soluble fiber, 8.3% ash) compared to the present study. However,

to truly understand AA digestibility, ileal digesta needs to be collected and corrected for diet specific

endogenous losses. As demonstrated by Johnson et al. (1998), nitrogen apparent total tract

digestibility is numerically higher than ileal digestibility, similar results were also reported by Murray

and others (1998) and Hendrix and Sritharan (2002). A precision-fed cecectomized rooster assay

reported (Oba and Swanson, 2019) that APS ileal digestibility was greater than 80% for most

indispensable AA, with some greater than 90% (Oba and Swanson, 2019); however, the authors did

not report which indispensable AA had digestibility lower than 80%.

Although some differences were observed over time, all the hematological parameters, with

one exception, were within reference range for this cohort of dogs (Table 4). Mean corpuscular

hemoglobin concentration (MCHC) was below reference range minimums on wk 0 and 13 (29.9

103/mm3 vs. 30.5 103/mm3, respectively; reference range: 31.0 g/dL – 39.0 g/dL), but within the

reference range on wk 26 (32.1 g/dL). While within reference range, alanine aminotransferase,

alkaline phosphatase, and glucose decreased over time (Table 5; P < 0.05). Despite some analyzed

parameters being affected over the course of the trial, it is unclear whether these changes have

biological significance. In the context of AAFCO regulatory support, the response in serum

biochemistry over 26 wk meet criteria for a complete and balanced adult maintenance claim.

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In conclusion, Labradors Retrievers fed a commercial grain-free diet had increased plasma

Met, cystine and taurine, and increased whole blood taurine concentrations over a 26-wk feeding

study. Urinary taurine:creatinine was not affected throughout the study and fecal excretion of BA

increased over time. The current study suggests that the grain-free diet tested does not affect

taurine status or gross indicators of health over a 26-wk period. Although dog’s cardiac function was

not evaluated in this study, it should be considered in future research.

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Table 1. Analyzed nutrient composition of kennel and test diets

Composition, % APS1 CTL2

Moisture 8.40 6.50

% dry matter

Crude protein 37.81 30.89

Crude fat 18.78 16.79

Ash 8.06 9.97

Nitrogen free extract3 23.95 28.66

Total dietary fiber 11.40 13.60

Insoluble fiber 9.50 11.90

Soluble fiber 1.90 1.70

Arginine 2.25 1.72

Histidine 0.79 0.55

Isoleucine 1.19 1.03

Leucine 2.31 2.32

Lysine 2.31 1.15

Methionine 0.55 0.43

Methionine+Cystine 0.85 0.99

Phenylalanine 1.32 1.20

Threonine 1.23 1.05

Valine 1.51 1.43

Tryptophan 0.30 0.22

Methionine:Cystine 1.83 0.77

Taurine 0.14 0.07

Cholesterol, mg/100g 140 98.5 1 APS: test diet, Acana port and squash. Ingredient composition: deboned pork, pork meal, whole

lentils, pork liver, pork fat, whole peas, lentil fiber, pea starch, butternut squash, pollock oil, natural

pork flavor, pork cartilage, pumpkin, salt, mixed tocopherols, zinc proteinate, dried kelp, calcium

pantothenate, taurine, freeze dried pork liver, copper proteinate, chicory root, turmeric, dried

Lactobacillus acidophilus fermentation product, dried Bifidobacterium animalis fermentation

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product, dried Lactobacillus casei fermentation product. Diet formulated to meet the requirement of

all life stages (AAFCO, 2018).

2 CTL: kennel diet, MFA Gold N Pro. Ingredient composition: poultry by-product meal, ground corn,

corn distillers dried grain with solubles, pearled barley, poultry fat, porcine meal, dried plain beet

pulp, poultry liver, flavors, flax seeds, and minerals and vitamins. Diet formulated to meet the

requirement of adult dogs at maintenance (AAFCO, 2018).

3 Nitrogen-free extract calculated as subtracting crude protein, crude fat, total dietary fiber, and ash

from the dry matter.

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Table 2. Whole blood taurine and plasma amino acid concentrations of Labrador Retrievers fed a

grain-free diet for 26 weeks (n = 8)

Amino acid, nmol/mL Week

SEM1 P-value 0 13 26

L-alanine 314b 267c 399a 15.8 <0.0001

L-arginine 66.8 82.3 63.0 29.5 0.8865

L-a-amino-n butyric acid 70b 125ab 193a 27.3 0.0159

L-asparagine 34.0 29.9 35.8 6.03 0.7838

L-aspartic acid 24.5b 33.9b 69.4a 9.26 0.0062

L-citrulline 30.4 26.1 29.3 11.1 0.9594

Cystathionine 33.5 60.8 66.4 13.1 0.1902

L-cystine 4.13 2.60 3.56 0.82 0.432

L-glutamic acid 46.5 24.0 23.2 15.2 0.482

L-glutamine 142 240 181 74.3 0.6485

Glycine 215b 595a 494a 55.3 0.0003

L-histidine 161b 185b 306a 35.9 0.0216

1-methyl-l-histidine 43.0 85.4 76.2 15.2 0.1412

3-methyl-l-histidine 8.3b 12.2ab 13.8a 1.41 0.0321

L-isoleucine 30.4 31.0 42.9 11.1 0.6758

L-leucine 75.4b 82.0b 144.0a 13.3 0.0025

L-lysine 114b 132b 234a 13.9 <0.0001

L-methionine 94b 128b 230a 24.0 0.0017

L-ornithine 38.8b 54.8b 86.6a 7.36 0.0006

L-phenylalanine 39.0b 39.7b 88.2a 9.91 0.0025

L-proline 90.8 90.8 138.5 18.1 0.123

Hydroxy-l-proline 72.8b 86.1b 251.4a 27.7 0.0002

L-serine 82.1 64.7 121.6 22.9 0.2243

Taurine 107c 157b 192a 11.4 0.0001

L-threonine 142ab 128b 199a 20.7 0.0557

Tryptophan 105b 169ab 226a 29.5 0.0291

L-tyrosine 39.4c 58.8b 73.4a 3.33 <0.0001

L-valine 99.0 81.2 122.1 27.9 0.5898

Taurine (whole blood) 186b 204b 295a 20.2 0.0021 abc Means in a row with different superscripts differ, P < 0.05.

1 SEM: standard error of the mean.

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Table 3. Urine taurine, creatinine, and taurine:creatinine ratio, and fecal bile acids and cholesterol of

Labrador Retrievers fed a grain-free diet for 26 weeks (n = 8)

Parameter Week

SEM1 P-value 0 26

Urine

Taurine: Creatinine 0.25 0.28 0.07 0.7760

Fecal Bile Acids, µg/mg

Cholic acid 0.055b 0.142a 0.024 0.0193

Chenodeoxycholic acid 0.008b 0.037a 0.008 0.0300

Lithocholic acid 0.74 0.94 0.07 0.0622

Deoxycholic acid 3.45b 5.52a 0.58 0.0237

Ursodeoxycholic acid 0.0065 0.0066 0.0004 0.8358

Primary bile acids 0.063b 0.179a 0.027 0.0078

Secondary bile acids 4.19b 6.46a 0.64 0.0255

Total bile acids 4.26b 6.64a 0.65 0.0208

Cholesterol 0.98 1.1 0.079 0.2860

Fecal Bile Acids, % of total

Cholic acid 1.33 2.2 0.37 0.1154

Chenodeoxycholic acid 0.18 0.73 0.19 0.0671

Lithocholic acid 18.08a 14.67b 1.05 0.0367

Deoxycholic acid 80.23 82.3 1.22 0.2515

Ursodeoxycholic acid 0.17a 0.11b 0.017 0.0204

Primary bile acids 1.51 2.93 0.51 0.0682

Secondary bile acids 98.49 97.07 0.51 0.0682 ab Means in a row with different superscripts differ, P < 0.05.


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Table 4. Hematology of Labrador Retrievers fed a grain-free diet for 26 weeks (n = 8)

Blood parameter Unit Week


WBC2 103/mm3 8.79b 10.33b 13.71a 1.15 0.0188

RBC4 106/mm3 8.24 8.19 8.47 0.15 0.379

Hemoglobin g/dL 16.3b 16.5ab 17.3a 0.31 0.0636

Hematocrit % 54.6 54.2 54.1 1.16 0.9573

MCV5 fl 66.4 66.3 63.8 1.05 0.1613

MCH6 pg 19.8b 20.2ab 20.4a 0.20 0.0721

MCHC7 g/dL 29.9b 30.5b 32.1a 0.34 0.0005

Platelets 103/mm3 265 314 308 38.2 0.6163

Lymphocytes 103/mm3 2.05a 1.53b 1.82ab 0.18 0.1382

% 23.1a 14.8b 14.5b 1.39 0.0003

Neutrophils 103/mm3 6.22b 7.89b 10.85a 0.93 0.007

% 71.0b 76.4a 78.7a 1.15 0.0003

Monocytes 103/mm3 0.36 0.43 0.63 0.13 0.3346

% 4.13 4.13 4.1 0.66 0.9995

Eosinophils 103/mm3 0.12b 0.38a 0.28ab 0.077 0.082

% 1.34b 3.65a 1.90b 0.56 0.0206

Basophils 103/mm3 0.040b 0.106a 0.095a 0.018 0.0403

% 0.44b 1.03a 0.71ab 0.15 0.033 ab Means in a row with different superscripts differ, P < 0.05.


2 WBC: white blood cell count.

4 RBC: red blood cell count.

5 MCV: mean corpuscular volume.

6 MCH: mean corpuscular hemoglobin.

7 MCHC: mean corpuscular hemoglobin concentration.

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Table 5. Blood chemistry of Labrador Retrievers fed a grain-free diet for 26 weeks (n = 8)

Blood parameter Unit Week


Total protein blood g/dL 6.29 6.36 6.25 0.13 0.8137

Albumin g/dL 3.84 3.88 3.7 0.082 0.3048

Globulin g/dL 2.44 2.46 2.56 0.15 0.8246

A/G ratio2

1.59 1.6 1.52 0.1 0.8429

ALT3 U/L 54.4a 38.1b 38.8b 4.28 0.0217

Alkaline phosphatase U/L 40.1a 28.6b 23.8b 2.87 0.0019

Total bilirubin mg/dL 0.30b 0.30b 0.36a 0.011 0.0004

Creatinine mg/dL 0.85b 0.84b 1.14a 0.053 0.0008

Blood urea nitrogen mg/dL 13.1c 17.8b 22.9a 1.34 0.0002

BUN/Creatinine ratio

15.5b 22.3a 20.2ab 1.74 0.0352

Phosphorus mg/dL 4.16b 4.81a 4.01b 0.17 0.0075

Glucose mg/dL 105a 103ab 98b 1.97 0.0655

Calcium mg/dL 10.6 10.5 10.5 0.085 0.5582

Sodium nmol/L 145ab 144b 146a 0.66 0.0597

Potassium nmol/L 4.13b 4.30ab 4.43a 0.085 0.0635 abc Means in a row with different superscripts differ, P < 0.05.


2 A/G ratio: albumin to globulin ratio.

3 ALT: alanine aminotransferase.

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R.A. Donadelli, J.G. Pezzali, P.M. Oba , C. Pendlebury ... · and Aldrich, 2019; Pezzali et al.,...

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Transcript of R.A. Donadelli, J.G. Pezzali, P.M. Oba , C. Pendlebury ... · and Aldrich, 2019; Pezzali et al.,...

R.A. Donadelli*, J.G. Pezzali*, P.M. Oba , C. Pendlebury ... · and Aldrich, 2019; Pezzali et al.,...

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Transcript of R.A. Donadelli*, J.G. Pezzali*, P.M. Oba , C. Pendlebury ... · and Aldrich, 2019; Pezzali et al.,...

R.A. Donadelli, J.G. Pezzali, P.M. Oba , C. Pendlebury ... · and Aldrich, 2019; Pezzali et al.,...

Transcript of R.A. Donadelli, J.G. Pezzali, P.M. Oba , C. Pendlebury ... · and Aldrich, 2019; Pezzali et al.,...